nmr detected hydrogen-deuterium exchange reveals differential dynamics of antibiotic and nucleotide...
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NMR Detected Hydrogen-Deuterium Exchange Reveals Differential Dynamics of Antibiotic and Nucleotide
Bound Aminoglycoside Phosphotransferase 3′-IIIa
Adrianne Norris
Department of Biochemistry, Cellular and Molecular Biology
Thesis Advisor: Dr. Engin Serpersu
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Introduction: Aminoglycoside Antibiotics
• Broad spectrum
• Meningitis
• Tuberculosis
• Diverse size/structure
+ ribosome
Translation Inhibited
Cell death
Aminoglycoside
Mechanism of Action
Kanamycins
Neomycins
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Introduction: Antibiotic Resistance
Enzyme catalyzed covalent modification
Aminoglycoside Phosphotransferase (3′)-IIIa (APH)
targets at least 10 different aminoglycosides of various size/structure
APH
ATP ADP
OPO3
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Research Goals
1) How is APH so promiscuous?
2) How is APH affected when interacting with different antibiotics?
Obtain a better understanding of protein-antibiotic interactions
More intelligent foundation for drug design to combat resistance
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No significant change in structure from apo to antibiotic bound?
Need structural information in solution to determine the mechanism of broad substrate selectivity – NMR!
How is APH so promiscuous?
Front Back
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How is APH so promiscuous?
NMR detected Hydrogen-Deuterium Exchange = In solution dynamics
Conclusion: Flexibility of APH allows modification of structurally diverse antibiotics.
Apo: H2O Apo: ~20hrs in D2O
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Apo-APH APH-Antibiotic
Suggests: A flexible apo-enzyme is the secret!
How is APH so promiscuous?
Nuclear Magnetic Resonance (NMR)
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How is APH affected when interacting with different antibiotics?
Little change in XL structures of APH-neomycin and APH-kanamycin complexes.
Kanamycin
Neomycin
Back
Front
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How is APH affected when interacting with different antibiotics?
Kanamycin
Neomycin
> 40 amino acids with different
environments
NMR
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Kanamycin Neomycin
Neomycin induces greater solvent protection of APH than kanamycin.
How is APH affected when interacting with different antibiotics?
NMR Hydrogen-Deuterium Exchange
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Green: Different Chemical Environment
Yellow: Different Solvent Exchange Properties
Conclusion: Neomycin Induces Greater Structural/Dynamic Stability than Kanamycin
How is APH affected when interacting with different antibiotics?
antibiotic
nucleotide
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Summary
• The broad substrate selectivity of APH is due to structural flexibility.
• Neomycin creates greater stability in APH than kanamycin
Future Directions• Neutron scattering experiments to determine differences in the radii of gyration of APH in various complexes – complementary to NMR
• Application of this type of analysis for AAC, aminoglycoside acetyltransferase
• Testing of synthetic inhibitor molecules.
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Acknowledgements
Dr. Engin Serpersu – Thesis advisor
Dr. Dan Roberts
Dr. Nitin Jain
Dr. David Baker
Dr. Jeremy Smith
Can Ozen
BCMB Department